Live images were taken every 24 h until 96 h to assess tdT and H2B-eGFP signals. Anti-TPPP3 antibodies were used to determine the maintenance of endogenous TPPP3 expression (Extended Data 6b ).
Tile scan images of each well were captured on the Leica TCS SP8 and the conversion index was calculated using automated filtering and thresholding in Imaris (Bitplane).
2) For multipotential differentiation assays, each sub-population was subjected to culturing and differentiation conditions per murine MSCs12 (link). murine MSCs (Invitrogen) were used as a positive control in parallel. For adipogenesis and chondrogenesis, cells were expanded in MSC growth media. For osteogenesis, cells were expanded in osteogenic growth media (αMEM, 20% MSC qualified FBS, 2 mM GlutaMax, 100 U/mL Penicillin-Streptomycin (Gibco), 10 nM Dexamethasone (Sigma)). At 80% confluency, cells were trypsinized, pelleted and seeded for assay.
Tile scan images of each well were captured on the Leica TCS SP8 and the conversion index was calculated using automated filtering and thresholding in Imaris (Bitplane).
2) For multipotential differentiation assays, each sub-population was subjected to culturing and differentiation conditions per murine MSCs12 (link). murine MSCs (Invitrogen) were used as a positive control in parallel. For adipogenesis and chondrogenesis, cells were expanded in MSC growth media. For osteogenesis, cells were expanded in osteogenic growth media (αMEM, 20% MSC qualified FBS, 2 mM GlutaMax, 100 U/mL Penicillin-Streptomycin (Gibco), 10 nM Dexamethasone (Sigma)). At 80% confluency, cells were trypsinized, pelleted and seeded for assay.